A great proportion of the activity and expenditure in the field of molecular biology is devoted to the isolation, modification, and expression of genes (sequences of nucleotides ranging from ˜400 bases to ˜several kilobases) and DNA constructs (e.g. gene systems, such as metabolic pathways and other long DNA constructs, ranging from several Kb to ˜300 Kb) for a range of applications, including the production of protein-based drugs, the production of chemicals and biofuels, and the creation of protein libraries, gene knockouts, and other mutations and modifications aimed at garnering a fundamental understanding of molecular biology.
Although it has been feasible for some time to synthesize small genes (<1 Kb) ab initio from synthetic oligonucleotides (i.e. sequences of DNA up to ˜100 nucleotide bases) [e.g. Gupta et al., Proc Natl Acad Sci, 57, 148 (1968); Stemmer et al., Gene, 164, 49 (1995)], the cost and error rate associated with this procedure has been limiting in terms of the size gene or the size of the gene library that can economically be synthesized. Recent advances in the use of DNA oligonucleotide microarray-based methods for synthesizing the synthetic oligonucleotide precursors [e.g. Tian et. al., Nature, 432, 1050 (2004); Zhou et al., Nucleic Acids Res, 32, 5409 (2004); Richmond et al., Nucleic Acids Res, 32, 5011], as well as error correcting methods [e.g. Carr et. al., Nucleic Acids Res, 32, e162 (2004); Tian et. al., Nature, 432, 1050 (2004)] for assembling such oligonucleotides into genes or larger DNA constructs, coupled with the vast sequence knowledge that has been accumulated through genome sequencing projects, have opened up the new possibility of ‘bit to gene’, in which a gene or other DNA construct can be downloaded or designed in silico and then directly synthesized. However, such procedures still involve macroscopic liquid volumes (typically greater than 1 microliter and as much as milliliters) and macroscopic fluid handling, which typically requires costly robotic handlers. In the case of microarray-derived oligonucleotide precursors, these precursors also require amplification, which introduces errors. Thus, the overall cost and time currently associated with synthesizing gene-length and longer DNA constructs (e.g. bacterial genomes) are prohibitive to widespread use and availability.
Court et al. have previously disclosed methods for inducing homologous combination using single-stranded nucleic acids [U.S. Pat. App. Pub. No. 2005/0079618; Court et al., Apr. 14, 2005] and, in Ellis et al., Proc Natl. Acad. Sci. USA 98:6742-6, 2001, single oligonucleotides have been employed to modify a genome, but these procedures do not solve these problems. What has been needed, therefore, is the ability to make several changes at once, so that gene-length and longer DNA constructs may be fabricated in a time and cost-effective manner.